A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells.

PURPOSE: The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. MATERIALS AND METHODS: Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. RESULTS: Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. CONCLUSIONS: This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

Radiation-induced cell cycle perturbations: a computational tool validated with flow-cytometry data.

Cell cycle progression can be studied with computational models that allow to describe and predict its perturbation by agents as ionizing radiation or drugs. Such models can then be integrated in tools for pre-clinical/clinical use, e.g. to optimize kinetically-based administration protocols of radiation therapy and chemotherapy. We present a deterministic compartmental model, specifically reproducing how cells that survive radiation exposure are distributed in the cell cycle as a function of dose and time after exposure. Model compartments represent the four cell-cycle phases, as a function of DNA content and time. A system of differential equations, whose parameters represent transition rates, division rate and DNA synthesis rate, describes the temporal evolution. Initial model inputs are data from unexposed cells in exponential growth. Perturbation is implemented as an alteration of model parameters that allows to best reproduce cell-cycle profiles post-irradiation. The model is validated with dedicated in vitro measurements on human lung fibroblasts (IMR90). Cells were irradiated with 2 and 5 Gy with a Varian 6 MV Clinac at IRCCS Maugeri. Flow cytometry analysis was performed at the RadBioPhys Laboratory (University of Pavia), obtaining cell percentages in each of the four phases in all studied conditions up to 72 h post-irradiation. Cells show early [Formula: see text]-phase block (increasing in duration as dose increases) and later [Formula: see text]-phase accumulation. For each condition, we identified the best sets of model parameters that lead to a good agreement between model and experimental data, varying transition rates from [Formula: see text]- to S- and from [Formula: see text]- to M-phase. This work offers a proof-of-concept validation of the new computational tool, opening to its future development and, in perspective, to its integration in a wider framework for clinical use.

Immunophenotyping Reveals No Significant Perturbation to PBMC Subsets When Co-cultured With Colorectal Adenocarcinoma Caco-2 Cells Exposed to X-Rays.

In vitro co-culture models between tumor cells and peripheral blood mononuclear cells (PBMCs) allow studying the interplay between these cell populations, potentially gaining insight into the in vivo response of the immune system to the presence of the tumor, as well as to possible other agents as radiation used for therapeutic purposes. However, great care is needed in the experimental optimization of models and choice of conditions, as some setups might offer a limited possibility to capture subtle immune perturbations. A co-culture model of PBMCs from healthy donors and colorectal adenocarcinoma Caco-2 cells was successfully adopted in a previous work to measure effects on Caco-2 and modulation of signaling when these latter are irradiated. We here tested if the same experimental setting allows to measure perturbations to the main PBMC subsets: we performed immunophenotyping by means of flow cytometry and quantified helper and cytotoxic T cells, NK cells, and B cells, when PBMCs are cultured alone (control), in presence of non-irradiated Caco-2 cells or when these latter are exposed to a 10 Gy X-ray dose from a conventional radiotherapy accelerator. To measure a baseline response in all experimental conditions, PBMCs were not further stimulated, but only followed in their time-evolution up to 72 h post-irradiation of Caco-2 and assembly of the co-culture. In this time interval PBMCs maintain a high viability (measured via the MTT assay). Caco-2 viability (MTT) is slightly affected by the presence of PBMCs and by the high radiation dose, confirming their radioresistance. Immunophenotyping results indicate a large inter-individual variability for different population subsets already at the control level. We analyzed relative population changes and we detected only a small but significant perturbation to cytotoxic T cells. We conclude that this model, as it is, is not adequate for the measurements of subtler immune perturbations (if any, not washed-out by inter-individual differences). For this purpose, the model needs to be modified and further optimized e.g., including a pre-treatment strategy for PBMCs. We also performed a pooled analysis of all experimental observations with principal component analysis, suggesting the potential of this tool to identify subpopulations of similarly-responding donors.

INVESTIGATION INTO THE PROBABILITY FOR MISCOUNTING IN FOCI-BASED ASSAYS.

When early radiation damage to biological systems is studied based on the formation of foci at the location of DNA double-strand breaks, the foci observed in irradiated cells either may be induced by ionizing radiation (IR) interactions or they may be due to other causes that lead to observation of foci also in unirradiated cells. Generally, to take account of the latter, additional samples are taken where the exposure to IR is skipped in the protocol. The data analysis relies on statistical independence of the frequency distributions of background and radiation-induced foci. In microscopy, however, the observed spatial patterns of foci are 2D projections of the spatial distributions of foci in the observed cell nuclei. This may lead to missing foci when scoring their number, particularly if projections of foci overlap or coincide. This paper investigates to what extent the statistical independence of the frequency distribution of the number of foci coming from IR interaction or other causes is compromised by foci overlapping.

MODELLING γ-H2AX FOCI INDUCTION TO MIMIC LIMITATIONS IN THE SCORING TECHNIQUE.

An approach based on track-structure calculations has been developed to take account of artefacts occurring during γ-H2AX foci detection in 2D images of samples analyzed through immunocytochemistry. The need of this works stems from the observed saturation in foci yields measured after X-ray doses higher than few grays, hindering an unambiguous quantification of DNA damage and of radiation effectiveness. The proposed modelling approach allows to simulate the observer's point of view for foci scoring, mimicking the selection of a slice Δz of the cell nucleus due to the microscope depth of field, and applying a clustering algorithm to group together damages within a resolution parameter r. Calculation results were benchmarked with experimental measurements at an early time-point for mouse breast cancer cells, irradiated with X-ray doses in the range 0-5 Gy. The model is able to reproduce the saturation in experimental data.

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum.

Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0-10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction scaffold protein expression levels, assumed to be related to IFN-γ- and TNF-α-mediated signaling.

In vitro engineering of human 3D chondrosarcoma: a preclinical model relevant for investigations of radiation quality impact.

BACKGROUND: The benefit of better ballistic and higher efficiency of carbon ions for cancer treatment (hadron-therapy) is asserted since decades, especially for unresectable or resistant tumors like sarcomas. However, hadron-therapy with carbon ions stays underused and raises some concerns about potential side effects for patients. Chondrosarcoma is a cartilaginous tumor, chemo- and radiation-resistant, that lacks reference models for basic and pre-clinical studies in radiation-biology. Most studies about cellular effects of ionizing radiation, including hadrons, were performed under growth conditions dramatically different from human homeostasis. Tridimensional in vitro models are a fair alternative to animal models to approach tissue and tumors microenvironment. METHODS: By using a collagen matrix, standardized culture conditions, physiological oxygen tension and a well defined chondrosarcoma cell line, we developed a pertinent in vitro 3D model for hadron-biology studies. Low- and high-Linear Energy Transfer (LET) ionizing radiations from GANIL facilities of ~1 keV/µm and 103 ± 4 keV/µm were used respectively, at 2 Gy single dose. The impact of radiation quality on chondrosarcoma cells cultivated in 3D was analyzed on cell death, cell proliferation and DNA repair. RESULTS: A fair distribution of chondrosarcoma cells was observed in the whole 3D scaffold. Moreover, LET distribution in depth, for ions, was calculated and found acceptable for radiation-biology studies using this kind of scaffold. No difference in cell toxicity was observed between low- and high-LET radiations but a higher rate of proliferation was displayed following high-LET irradiation. Furthermore, 3D models presented a higher and longer induction of H2AX phosphorylation after 2 Gy of high-LET compared to low-LET radiations. CONCLUSIONS: The presented results show the feasibility and usefulness of our 3D chondrosarcoma model in the study of the impact of radiation quality on cell fate. The observed changes in our tissue-like model after ionizing radiation exposure may explain some discrepancies between radiation-biology studies and clinical data.